(A) Schematic for analysis of conidial A-PCD markers. NF, neutrophils; AF, Aspergillus fumigatus. (B) Murine lung CD45– cells and CD45+CD11b+Ly6G+ neutrophils analyzed for RFP and AF633 fluorescence 24 hpi with 3 × 107 AF633+H-RFP conidia. (C to G) Free conidia [orange gate in (B), lines in (D) and (E), and bars in (F) and (G)] and live [red gate in (B), lines in (D) and (E), and bars in (F) and (G)] and nonviable [blue gate in (B), lines in (D) and (E), and bars in (F) and (G)] conidia internalized in neutrophils were analyzed by (C) fluorescence microscopy (scale bar, 20 μm) and [(D) to (F)] flow cytometry for [(C) and (D)] caspase activity and [(E) and (F)] DNA fragmentation by TUNEL and (G) for fungal viability. [(F) and (G)] The data are presented as mean + SD of three independent experiments, each performed with triplicate samples. Statistical analysis: Kruskal-Wallis rank sum test, followed by a Dunn’s test for multiple comparisons.

To investigate its relevance for pathogenesis, we engineered A. fumigatus strains that are modified in their response to A-PCD induction. Using a bioinformatics approach, we identified AfBIR1 (hereafter referred to as BIR1), a homolog of human survivin (11) and Saccharomyces cerevisiae BIR1 (12) (Fig. 2A and fig. S2A), both inhibitor-of-apoptosis protein family members (13). Survivin encodes a single BIR domain and suppresses apoptosis by inhibiting caspase-3 and -7 (14). Transgenic strains that overexpress full-length A. fumigatus BIR1 (BIR1OX1,2) (Fig. 2B and fig. S2, B and C) exhibited a similar radial growth, conidiation, and germination rate as the parental H-RFP strain (fig. S2, D to F). In both full-length BIR1OX isolates, we observed reduced A-PCD compared with the parental strain under oxidative stress in vitro, as judged by reduced RFP signal loss, reduced fungal caspase activity, reduced number of TUNEL-positive nuclei, and enhanced survival (Fig. 2, C to F). Overexpression of the BIR1 N-terminal region (N-BIR1OX1,2) (Fig. 2, A and B, and fig. S2, A to C) that includes two BIR domains was sufficient to recapitulate the phenotypes observed in isolates that overexpress the full-length gene (Fig. 2, C to F). In two independent attempts, we did not succeed in generating a bir1 null strain. Placement of the BIR1 gene under control of a tetracycline-inducible promoter (i.e., bir1tetON strain) indicated that the gene is essential for growth under laboratory conditions tested (fig. S3). In the absence of a bir1 null strain, we added S12 (15), a Survivin antagonist that targets the BIR domain, and observed more intense TUNEL staining in H-RFP compared with BIR1OX1 fungal cells (Fig. 2G and fig. S4). Moreover, S12 sensitized fungal cells to subapoptotic H2O2 levels, with more pronounced effects on H-RFP than on BIR1OX and N-BIR1OX fungal cells, as judged by reduced RFP fluorescence, increased fungal caspase activity, increased TUNEL signal, and reduced fungal viability. The analysis of four independent isolates with similar phenotypes and the use of a pharmacologic inhibitor preclude the likelihood of off-target effects of the overexpression strategy. Collectively, these data indicate that BIR1 mediates anti-PCD activity in A. fumigatus; the BIR1 N-terminal region is sufficient for this function.

Consistent with these findings, there was a transient increase in total lung leukocytes, neutrophils, and inflammatory monocytes in BIR1OX1-challenged mice compared with H-RFP–challenged mice 24 hours postinfection (hpi) (fig. S6A), although differences resolved by 48 hpi. The number of lung monocyte–derived dendritic cells (Mo-DCs) was higher in the BIR1OX1-challenged group at 48 hpi, consistent with the developmental relationship between infiltrating monocytes and Mo-DCs (16). Challenge with BIR1OX1 conidia resulted in higher lung inflammatory cytokine [tumor necrosis factor, interleukin-1α (IL-1α), and IL-1β] and chemokine (CXCL1, CXCL2, and CXCL5) levels at 24 hpi than challenge with H-RFP conidia (fig. S6B). Because irradiated H-RFP and BIR1OX1 germlings induced equivalent macrophage inflammatory responses in vitro (fig. S2G), the higher level of tissue inflammation in BIR1OX1-challenged mice reflected a higher fungal burden rather than a strain-specific difference in the induction of host inflammation. Collectively, these data indicate that the BIR1OX1 strain is more virulent than the H-RFP strain and leads to invasive aspergillosis in immune-competent mice.

To determine the mechanism by which BIR1 expression levels promote invasive aspergillosis, we compared leukocyte conidial uptake and killing in BIR1OX1- and H-RFP–challenged mice (Fig. 3F). The frequency of lung neutrophil conidial uptake (Fig. 3G) was slightly higher for BIR1OX1 than for H-RFP conidia at 24 hpi, but this finding was not observed for airway neutrophils, lung monocytes, or Mo-DCs, suggesting that differences in conidial uptake are unlikely to account for the difference in fungal virulence. In contrast, the frequency of fungus-engaged lung and airway neutrophils that contain live conidia was higher by a factor of 1.7 (23.7 ± 2.0% versus 14.3 ± 1.7%) and 2 (35.3 ± 4.5% versus 17.7 ± 4.3%) in BIR1OX1-challenged than in H-RFP–challenged mice, respectively, at 24 hpi (Fig. 3H). Similarly, the frequency of fungus-engaged lung monocytes and Mo-DCs that contain live BIR1OX1 conidia was higher by a factor of 1.57 (51.8 ± 3.8% versus 33.0 ± 4.1%) and 1.72 (15.9 ± 1.0% versus 9.3 ± 1.0%) in BIR1OX1-challenged than in H-RFP–challenged mice, respectively. These data indicate that BIR1 overexpression confers protection against myeloid cell–mediated conidial killing in the lung. To exclude the possibility that differences in neutrophil influx contribute to these results, we examined BIR1OX1,2, N-BIR1OX1,2, and H-RFP conidial uptake by and survival in murine (fig. S7, A and B) and human neutrophils ex vivo (Fig. 3, I to L). BIR1OX1,2 and N-BIR1OX1,2 conidial viability in fungus-engaged human and murine neutrophils was higher than that of H-RFP conidia (Fig. 3J and fig. S7C), and the frequency of caspase+ and TUNEL+ fungal cells was much lower for the BIR1OX1,2 or N-BIR1OX1,2 strains compared with the H-RFP strain (Fig. 3, K and L; fig. S7D), even when hyphae were coincubated with human neutrophils (fig. S8).

To determine host mechanisms that induce A. fumigatus A-PCD, we compared BIR1OX1 and H-RFP growth under a variety of stress conditions (fig. S9). Both strains exhibited similar growth under acidic and basic conditions and in the presence of cell wall–perturbing agents and sodium nitrite. However, the BIR1OX1 strain displayed a growth advantage under oxidative stress, including menadione and H2O2. This finding suggested that resistance of BIR1OX1 conidia to myeloid cell killing is likely related to induction of A-PCD by phagocyte NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase (i.e., NOX2). Humans with chronic granulomatous disease have genetic defects in NOX2 and a 40% lifetime risk of invasive aspergillosis (17), highlighting the role of NOX2 in barrier immunity against ubiquitous A. fumigatus exposure. To examine this hypothesis, we measured BIR1OX1 and H-RFP conidial killing in mixed bone marrow chimeric mice that contain both NADPH oxidase-sufficient and -deficient neutrophils in the lung (Fig. 4F). BIR1OX1 conidia survived equally well in p91phox−/− (28.6 ± 3.5%) and in p91phox+/+ neutrophils (29.3 ± 2.6%). In contrast, H-RFP conidia exhibited 32.6 ± 3.5% viability in p91phox−/− neutrophils but only 17.8 ± 2.7% viability in p91phox+/+ neutrophils (Fig. 4, F and G), indicating that BIR1 expression levels can counter the fungicidal activity of neutrophil NADPH oxidase by raising the threshold for A-PCD induction.

To investigate the conjecture that the difference in virulence between BIR1OX1 and H-RFP conidia depends on host NADPH oxidase expression, we challenged p91phox−/− mice with 5 × 104BIR1OX1 or H-RFP conidia. In contrast to p91phox+/+ mice (Fig. 3A and fig. S5), the survival of p91phox−/− mice was similar whether BIR1OX1 or H-RFP conidia were administered (Fig. 4H). These data show that the virulence of the BIR1OX1 strain is comparable to that of the parental strain in the absence of host NADPH oxidase. Our results advance the concept that sterilizing immunity against mold conidia exploits fungal A-PCD to prevent the formation of multicellular, tissue-invasive hyphae in the lung (fig. S10). In this model, pathogen virulence is a product of microbial (i.e., fungal Bir1p) and host factors (i.e., NADPH oxidase) in determining disease development (18). Although fungi exhibit a conserved set of apoptotic markers (19), the fungal A-PCD apparatus is notably different from the mammalian apoptotic apparatus (20). Targeted pharmacologic blockade of key components in the fungal anti-PCD response, exemplified by Bir1p, may augment prophylactic or therapeutic approaches against invasive aspergillosis and highlight the importance of characterizing differences in protein structure between mammalian and fungal BIR domains involved in PCD. Identification of additional host effectors and compounds (21) that induce A-PCD in conidia and hyphae may inform new strategies for therapeutic intervention in vulnerable patient groups.